<i>LncRNA-HIT</i> Functions as an Epigenetic Regulator of Chondrogenesis through Its Recruitment of p100/CBP Complexes

<div><p>Gene expression profiling in E 11 mouse embryos identified high expression of the long noncoding RNA (lncRNA), <i>LNCRNA-HIT</i> in the undifferentiated limb mesenchyme, gut, and developing genital tubercle. In the limb mesenchyme, <i>LncRNA-HIT</i> was found to be retained in the nucleus, forming a complex with p100 and CBP. Analysis of the genome-wide distribution of <i>LncRNA-HIT</i>-p100/CBP complexes by ChIRP-seq revealed <i>LncRNA-HIT</i> associated peaks at multiple loci in the murine genome. Ontological analysis of the genes contacted by <i>LncRNA-HIT-</i>p100/CBP complexes indicate a primary role for these loci in chondrogenic differentiation. Functional analysis using siRNA-mediated reductions in <i>LncRNA-HIT</i> or p100 transcripts revealed a significant decrease in expression of many of the <i>LncRNA-HIT</i>-associated loci. <i>LncRNA-HIT</i> siRNA treatments also impacted the ability of the limb mesenchyme to form cartilage, reducing mesenchymal cell condensation and the formation of cartilage nodules. Mechanistically the <i>LncRNA-HIT</i> siRNA treatments impacted pro-chondrogenic gene expression by reducing H3K27ac or p100 activity, confirming that <i>LncRNA-HIT</i> is essential for chondrogenic differentiation in the limb mesenchyme. Taken together, these findings reveal a fundamental epigenetic mechanism functioning during early limb development, using <i>LncRNA-HIT</i> and its associated proteins to promote the expression of multiple genes whose products are necessary for the formation of cartilage.</p></div

Clinicopathologic features of primary breast carcinoma from breast tissue microarray.

<p>Clinicopathologic features of primary breast carcinoma from breast tissue microarray.</p

Strong EZH2 and <i>HOTAIR</i>

<p><b>coexpression trend with worse survival.</b> Kaplan-Meier curve of overall survival based upon the primary carcinoma expressions of both EZH2 and <i>HOTAIR</i> stratified into five different groups: both EZH2 and <i>HOTAIR</i> negative scores (orange line), one negative and one positive score (black line), both EZH2 and <i>HOTAIR</i> weakly positive scores (green line), one weak positive and one strong positive score (blue line), and both EZH2 and <i>HOTAIR</i> strongly positive scores (pink line). The number of cases in each group is listed. As the expression of both EZH2 and <i>HOTAIR</i> become positive and stronger, the percent survival tends to decrease. Strong expression of both EZH2 and <i>HOTAIR</i> together correlate with a trend toward worse survival (logrank test for trend p-value 0.0739).</p

Structure and Thermodynamics of N⁶‑Methyladenosine in RNA: A Spring-Loaded Base Modification

N⁶-Methyladenosine (m⁶A) modification is hypothesized to control processes such as RNA degradation, localization, and splicing. However, the molecular mechanisms by which this occurs are unclear. Here, we measured structures of an RNA duplex containing m⁶A in the GGACU consensus, along with an unmodified RNA control, by 2D NMR. The data show that m⁶A–U pairing in the double-stranded context is accompanied by the methylamino group rotating from its energetically preferred <i>syn</i> geometry on the Watson–Crick face to the higher-energy <i>anti</i> conformation, positioning the methyl group in the major groove. Thermodynamic measurements of m⁶A in duplexes reveal that it is destabilizing by 0.5–1.7 kcal/mol. In contrast, we show that m⁶A in unpaired positions base stacks considerably more strongly than the unmodified base, adding substantial stabilization in single-stranded locations. Transcriptome-wide nuclease mapping of methylated RNA secondary structure from human cells reveals a structural transition at methylated adenosines, with a tendency to single-stranded structure adjacent to the modified base

Co-recruitment of <i>LncRNA-HIT</i> and p100 is required to stimulate gene expression from a synthetic locus.

<p><b>(A)</b> Analysis of UAS-luciferase reporter activation after GAL4-λN-BoxB<i>LncRNA-HIT</i> and GAL4-p100 recruitment. <b>(B)</b> Analysis of UAS-luciferase reporter activation in the absence of GAL4-p100. <b>(C)</b> Model of UAS-luciferase reporter activation in response to the recruitment of <i>LncRNA-HIT</i> and p100. For panels A and B, increasing dosages (0, 50, 100, 250, ng) of the BoxB or BoxB-<i>LncRNA-HIT</i> transcripts are represented by the black triangle.</p

Expression of <i>HOTAIR</i> and EZH2 in primary versus metastatic carcinoma.

<p>Primary breast carcinomas with their matched metastases are stained with an RNA in situ hybridization probe for <i>HOTAIR</i> (rows 1 and 2) or immunohistochemical antibody for EZH2 (rows 3 and 4). Expression levels are indicated as negative (−), weakly positive (+), and strongly positive (++). As depicted, some metastatic carcinomas have increased expression compared to their matched primary carcinomas, while some metastatic carcinomas have decreased expression compared to their matched primary carcinomas.</p

<i>LncRNA-HIT</i> recruits a complex of p100 and CBP in the limb.

<p><b>(A-D)</b><i>Snd1</i> which encodes p100 is expressed in many of the same regions as <i>LncRNA-HIT</i> including the limbs at E11-13.5, spinal cord, and genital tubercle. FL = forelimb, HL = hindlimb, MX = maxillary component of the first branchial arch, SC = spinal cord, P = pinnae. <b>(E)</b> Western blot analysis using antibodies specific for CBP and p100 confirm the presence of both proteins in cell lysates from E 11.5 limb buds. <b>(F)</b> Streptavidin-mediated precipitation of biotinylated <i>LncRNA-HIT</i> and <i>U1</i> RNA transcripts reveals co-precipitation of CBP and p100 from limb mesenchyme, confirming recruitment of both proteins by the lncRNA in the limb mesenchyme. <b>(G)</b> RIP analysis of p100 and CBP from the limb mesenchyme reveals co-precipitation of endogenous <i>LncRNA-HIT</i> transcript, confirming that CBP, p100, and <i>LncRNA-HIT</i> are present in a complex in the limb bud mesenchyme. Values represent the mean fold enrichment of <i>LncRNA-HIT</i> after precipitation with antibodies specific for CBP, p100 from three independent assays. Control precipitations were performed in parallel using murine IgG. Bars represent the standard deviation of the mean from the three independent assays.</p

Expression analysis of the lncRNA <i>LncRNA-HIT</i>.

<p><b>(A)</b><i>In situ</i> hybridization using an antisense <i>LncRNA-HIT</i> riboprobe detects the transcript as early as E 10.5 in the distal limb which expands throughout the limb bud at E 11.5 <b>(B)</b>. <b>(C)</b> <i>In situ</i> hybridization using a sense orientation <i>LncRNA-HIT</i> riboprobe detects no <i>LncRNA-HIT</i> transcripts, confirming the unidirectional transcription of <i>LncRNA-HIT</i> in the same orientation as the 5’ HoxA genes in the developing limb. <b>(D and E)</b> Analysis of <i>Hoxa13</i> expression shows a high level of overlap with <i>LncRNA-HIT</i> in the distal limb. <b>(F-H)</b> Analysis of <i>Hoxa9-Hoxa11</i> expression in the E 11.5 distal limb reveals some overlap with <i>LncRNA-HIT</i> in the limb bud. <b>(I-K)</b> Analysis of <i>Hoxd11-Hoxd13</i> expression in the E 11.5 distal limb reveals some overlap with <i>LncRNA-HIT</i>. <b>(L)</b> <i>LncRNA-HIT</i> is also expressed in the developing genital tubercle, gut epithelium, urogenital sinus, spinal cord, and vertebral bodies at E 13.5. <b>(M-N)</b> <i>LncRNA-HIT</i> expression is detected in the digit perichondrial tissues, digit joint fields, and in the developing carpal/tarsal skeletal elements in E 13.5 forelimbs and hindlimbs. <b>(O)</b> Relative fold expression of <i>LncRNA-HIT</i> and <i>Hottip</i> expression in the E 11.5 limbs as determined by qRTPCR. Values represent the <i>Gapdh</i> normalized average expression of <i>LncRNA-HIT</i> and Hottip in the limb calculated from three independent analyses. Bars represent the standard deviation of the mean from the three independent assays. UGS = urogenital sinus, VB = vertebral body, GT = genital tubercle, GE = gut epithelium, SC = spinal cord.</p

siRNA-mediated reduction in <i>LncRNA-HIT</i> or <i>Snd1</i> results in reduced levels of 5’ HoxA gene expression.

<p><b>(A)</b> Relative gene expression after transfection with <i>LncRNA-HIT</i> siRNAs or scrambled control siRNAs. Values represent average expression levels calculated from six independent replicates. Bars represent the standard deviation of the mean from the six independent replicates. Asterisks denote a significant changes in gene expression as determined a Student’s t test. <b>(B)</b> Relative gene expression after transfection with <i>Snd1</i> siRNAs or scrambled control siRNAs. Values represent average expression levels calculated from six independent replicates. Bars represent the standard deviation of the mean from the six independent replicates. Asterisks denote a significant changes in gene expression as determined a Student’s t test.</p

Model of enhancer RNA function of <i>LncRNA-HIT</i> at the 5’ HoxA locus.

<p>Model of enhancer RNA function of <i>LncRNA-HIT</i> at the 5’ HoxA locus.</p